Background: Carotid intima-media thickening (IMT) is a major risk factor for cardiovascular disease (CVD). The large ribosomal subunit protein 17 (Rpl17) was recently reported as a CVD-associated gene; however, ribosomal mutations generally are not associated with vascular dysfunction. We have created a novel genetic model of decreased ribosomal protein in endothelial cells (EC) to determine how changes in endothelial ribosomes lead to IMT as a predictive risk factor for CVD. Methods: EC-restricted RpL17 heterozygous mice (Cdh5-Cre; RpL17fl/wt, or Rpl17-Het), were generated and subjected to sham or partial carotid ligation (PCL) surgery of the left artery to induce acute disturbed (d)-flow in vivo. Carotids were harvested on day 14 for quantitative tissue immunostaining. Purified mouse and human EC in vitro were exposed to steady (s)-flow or d-flow using cone viscometry, and collected for flow cytometry, protein expression, electron microscopy, or ribosomal fractions. Human carotid samples from healthy and endarterectomy patients were used for tissue analysis. Results: Carotids from RpL17-Het mice with PCL-induced d-flow showed increased IMT relative to RpL17-WT controls. In addition, RpL17 protein levels were decreased in regions of d-flow compared to s-flow. Increased levels of ER stress markers were observed by carotid immunostaining, as well as activation of the integrated stress response (ISR) in RpL17-Het EC. Analysis of mRNAs bound to polysomes vs. monosomes in EC-RpL17-Het revealed increased translational efficiency of key regulators of glycolysis, redox, inflammation, matrix, and endothelial-to-mesenchymal transition (EndMT). Metabolic profiling by Seahorse assay confirmed enhanced anaerobic glycolysis and decreased oxidative respiration in RpL17-Het EC, and immunostaining of carotids identified upregulated EC inflammation and EndMT regulators. Conclusions: We have identified a novel pathway for d-flow-mediated IMT: d-flow ER stress (from decreased RpL17) ISR with altered protein translation EC metabolic reprogramming EC inflammation, proliferation, and EndMT IMT. These results establish flow pattern as a key biomechanical stimulus for RpL17-dependent endothelial cell phenotypic modulation. These data link a human CAD-associated ribosomal gene with disturbed flow to define a spatially restricted pathway for IMT. Key words: ribosomal protein 17 (RpL17), intima-media-thickening, shear stress, endothelial cell, translation
Wines-Samuelson, M., Chowdhury, S., Senchanthisai, S., Shaposhnikov, M., Sowden, M., Berk, B. C.
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